WO2019078606A2 - Composition anti-inflammatoire, antiallergène et améliorant la dermatite atopique utilisant un extrait de sargassum horneri ou un extrait d'ecklonia veine - Google Patents

Composition anti-inflammatoire, antiallergène et améliorant la dermatite atopique utilisant un extrait de sargassum horneri ou un extrait d'ecklonia veine Download PDF

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WO2019078606A2
WO2019078606A2 PCT/KR2018/012234 KR2018012234W WO2019078606A2 WO 2019078606 A2 WO2019078606 A2 WO 2019078606A2 KR 2018012234 W KR2018012234 W KR 2018012234W WO 2019078606 A2 WO2019078606 A2 WO 2019078606A2
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extract
composition
mixture
atopic dermatitis
allergic
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PCT/KR2018/012234
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Korean (ko)
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WO2019078606A3 (fr
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안긴내
지영흔
전유진
강나래
김서영
조진희
한의정
김현수
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전남대학교산학협력단
제주대학교 산학협력단
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Priority claimed from KR1020170135223A external-priority patent/KR20190043317A/ko
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Publication of WO2019078606A2 publication Critical patent/WO2019078606A2/fr
Publication of WO2019078606A3 publication Critical patent/WO2019078606A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus

Definitions

  • the present invention relates to an anti-inflammatory composition, an anti-allergic composition and a composition for improving atopic dermatitis using a Sargassum horneri extract or Ecklonia cava extract.
  • Atopic dermatitis is a recurrent chronic skin disease that occurs frequently in infancy, but it may persist in adults or develop into an adult, and its prevalence is increasing recently (Kor J Pharmacogn., 43: 59-65, 2012 ).
  • Atopic dermatitis is a type of allergic disease characterized by symptoms such as dry skin, itching, irritation, pruritus, swelling around the hair follicle, hypophytosis, ecchymosis, and eczema lesions (Korean J Invest Dermatol., 14: 67-72 , 2007).
  • the immune system a defense mechanism against external invasion of the human body, is centered on the activation of T cells.
  • the cytokines IL-2, IFN- ⁇ and TNF produced by Th1 cells induce the differentiation of Th1 cells and suppress the proliferation and differentiation of Th2 cells
  • IL-4, IL-5, IL Cytokines such as IL-6, IL-10, IL-13 and TSLP (thymic stromal lymphopoietin) induce the proliferation and differentiation of Th2 cells and inhibit the differentiation of Th1 cells (J Am Acad Dermatol., 44, S1- 2001). Under normal conditions, the interaction between Th1 and Th2 cells is balanced by maintaining the immune response.
  • Atopic dermatitis promotes the conversion of T cells into Th2 cells, and increased Th2 cells secrete a large amount of cytokines to produce IgE (J Clin Invest. 113 (5): 651-7, 2004; J Allergy Clin Immunol. 2003; 112 (2)), which induces hypersensitive responses by inducing mast cell and eosinophil differentiation : 252-62; Kor J Pharmacogn, 43: 59-65, 2012).
  • TSLP is overexpressed in skin lesions in patients with atopic dermatitis and overexpression of TSLP by keratinocyte in mice has been reported to cause atopic dermatitis (Nature immunology. 2002; 3 (7): 673-80 ; Methods in enzymology. 2014; 535: 371-87; The Journal of experimental medicine. 2005; 202 (4): 541-9).
  • Chemokine is a low-molecular protein that displays chemoattractants and is divided into four types according to its structure and function: C, CC, CXC, and CX3C. Recently, these chemokines have been implicated in the development and maturation of immune cells, And migration, Th1 / Th2 balance, and so on.
  • TARC Thimus & activation-regulated chemokine
  • CCR4 CC chemokine receptor 4
  • atopic dermatitis presents symptoms such as folliculitis, scurvy and dryness.
  • a feeling of pruritus occurs throughout the acute phase and the chronic phase, which is important in the treatment of atopic dermatitis because it causes secondary skin symptoms such as wound infection and secondary infection due to scratching.
  • the skin stimulation model tends to be preferred.
  • the skin irritation model is a method of inducing atopic dermatitis using chemical (DNCB: dinitrofluorobenzene, DNFB: dinitrofluorobenzene, PiCl: picryl chloride), which can induce dermatitis in a relatively short period of time with acute atopy induction model, It is useful for research because it has the merit of being able to visually confirm the wind and wind blasts.
  • DNCB dinitrofluorobenzene
  • DNFB dinitrofluorobenzene
  • PiCl picryl chloride
  • non-steroids also have a variety of side effects such as erythema, itching, edema, erythematosus, and hypoglycemia and weakness of immunity, making it difficult to treat essential atopic dermatitis (Arellano FM et al., J Invest Dermatol 2007 Apr ; 127 (4): 808-16.). Therefore, research is needed to find a substance with superior therapeutic effects from relatively safe natural products.
  • the present invention relates to a method for inhibiting the proliferation and the proliferation of Houttuynia cordata, a kind of seaweed, which has anti-inflammatory activity and antiallergic activity in cell experiments, It was confirmed that it has atopic dermatitis improving activity.
  • the present invention suppresses NO production in a concentration-dependent manner in mouse macrophage cells (RAW 264.7 cells) stimulated with LPS (lipopolysaccharide), as shown in the following examples and experimental examples, (Anti-DNP-IgE) and antigens (DNP-BSA), as well as anti-inflammatory activity by inhibiting the production of inflammatory cytokines (IL-1 ⁇ , IL-6 and TNF- Bone marrow-derived obesity cell line BMCMCs exhibit antihistamine activity and antiallergic activity, such as inhibiting degranulation.
  • LPS lipopolysaccharide
  • the present invention can be understood as an anti-inflammatory composition, an anti-allergic composition, and a composition for improving atopic dermatitis, which comprises an extract of Horns mellifera, a moxibustion extract, or a mixture thereof as an active ingredient.
  • hoe saengmyong mackerel or mackerel extract means water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.) Methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, Extracts obtained by leaching using a mixed solvent of carbon dioxide and pentane, or fractions obtained by fractionating the extracts obtained by using a supercritical extraction solvent such as pentane, Any method such as cold rolling, refluxing, heating, ultrasonic irradiation, supercritical extraction, etc.
  • the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying and the like.
  • an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent and more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.
  • active ingredient alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which itself is not active.
  • anti-inflammatory is meant to include improvement of an inflammatory disease (alleviation of symptoms), treatment, inhibition or delay of onset of such a disease as defined below.
  • inflammatory disease means an inflammatory reaction specified by an external physical or chemical stimulus or infection of an external infectious source such as bacteria, fungus, virus, various allergenic substances, or local or systemic defense against autoimmunity which may be defined as a pathological symptom.
  • an external infectious source such as bacteria, fungus, virus, various allergenic substances, or local or systemic defense against autoimmunity
  • inflammatory responses are caused by activation of various inflammatory mediators and enzymes associated with immune cells (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-a, IL-6, Cell migration, and tissue destruction, and manifest externally by symptoms such as erythema, pain, edema, fever, loss or loss of specific function of the body.
  • the inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so that as long as any disease is included in the definition of inflammatory diseases as described above, it may be acute, chronic, ulcerative, Whether it is necrotic or not.
  • the inflammatory diseases include inflammatory diseases such as asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, Inflammatory bowel syndrome, inflammatory pain, migraine headache, headache, back pain, fibromyalgia, fascia disease, viral infection (e.g., C type infection), bacterial infection, fungal infection, burn, wound due to surgical or dental surgery, Rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, ulceris, scleritis, uveitis, dermatitis (
  • antiallergic is meant to include improvement (symptom relief), treatment, prevention (prevention or delay of onset) of allergic diseases defined below.
  • allergic disease means pathological symptoms caused by allergic reactions mediated by mast cell activation such as degranulation of mast cells.
  • allergic diseases include atopic dermatitis, allergic asthma asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, allergic keratitis, and the like.
  • atopic dermatitis is defined to include all diseases classified as atopic dermatitis in the art accompanied by a skin irritable immune response, regardless of the cause, either directly or indirectly.
  • atopic dermatitis is classified into infantile atopic dermatitis, infantile atopic dermatitis, adult atopic dermatitis and maternal atopic dermatitis according to the onset period or the object of the invention.
  • atopic dermatitis includes all these types of atopic dermatitis .
  • improvement of atopic dermatitis means treatment of atopic dermatitis, prevention and improvement (alleviation of symptoms).
  • composition of the present invention may contain any amount (effective amount) of the active ingredient as long as it can exhibit anti-inflammatory activity, antiallergic activity, and atopic dermatitis improving activity which are intended to be treated according to the use, formulation, A typical effective amount will be determined within the range of from 0.001% to 15% by weight based on the total weight of the composition.
  • effective amount refers to an amount of an effective amount of the composition of the present invention, which is administered to a mammal, preferably a human, refers to the amount of the active ingredient contained in the composition of the present invention, which can exhibit antiphlogistic effects. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.
  • composition of the present invention may further contain, in addition to the active ingredient, any compound or natural extract known to have anti-inflammatory activity, anti-allergic activity, anti-allergic activity, can do.
  • Such compounds or extracts include dry powder of Enterococcus faecalis which is individually recognized as a function of 'overexpression of immune response' according to the Act on Health Functional Foods, complexes such as guava leaf extract, extract of Quercus mongolica extract, L-sakei Probio 65, a combination of PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) Lactic acid bacteria ( L. plantarum CJLP133), probiotic ATP, and the like.
  • compositions of the present invention may be included in the compositions of the present invention in combination with one or more of their active ingredients.
  • the composition of the present invention can be identified as a food composition.
  • its use can be understood as suppression of skin hypersensitivity.
  • the food composition of the present invention can be produced in any form and can be used in various forms such as beverages such as tea, juice, carbonated drink, ionic drink, processed oil such as milk and yogurt, gum, rice cake, Korean confectionery, A food, a health food, a food, a tablet, a capsule, a ring, a granule, a liquid, a powder, a slice, a paste, a syrup, a gel, a jelly and a bar.
  • beverages such as tea, juice, carbonated drink, ionic drink, processed oil such as milk and yogurt, gum, rice cake, Korean confectionery, A food, a health food, a food, a tablet, a capsule, a ring, a granule, a liquid, a powder, a slice, a paste, a syrup, a gel, a jelly and a bar.
  • the food composition of the present invention may be classified into any product category as long as it meets the laws and regulations on the time of manufacture and distribution in the legal and functional category.
  • it is a health functional food according to the ⁇ Health Functional Food Act ⁇ in Korea, or a food functional food according to the Korean Food Sanitation Law (Food Standards and Specifications) , Special-purpose food, and the like.
  • the food composition of the present invention may contain food additives in addition to the active ingredients thereof.
  • Food additives are generally understood to be substances that are added to foods and mixed or infiltrated into food in the manufacture, processing or preservation of food, and their safety must be ensured since they are ingested daily with food and for long periods of time.
  • Food additives according to the laws of the respective countries (“Food Sanitation Act” in Korea) regulating the manufacture and distribution of food, food additives with safety are specified in terms of ingredient or function.
  • Food Additives Code of Korea Food Additives Standards and Standards
  • Such food additives are classified into sweeteners, flavors Preservatives, emulsifiers, acidulants, and thickeners.
  • the sweetener is used for imparting a sweet taste suitable for foods, and both natural and synthetic sweeteners can be used in the composition of the present invention.
  • natural sweeteners are used.
  • natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.
  • Flavors may be used to enhance taste or flavor, both natural and synthetic.
  • a natural one is used.
  • natural ones the purpose of nutritional fortification can be performed in addition to the flavor.
  • natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine.
  • ginseng red ginseng
  • bamboo shoots, aloe vera, banks and the like can be used.
  • the natural flavoring agent may be a liquid concentrate or a solid form of extract.
  • Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.
  • calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA ethylenediaminetetraacetic acid
  • emulsifier acacia gum, carboxymethyl cellulose, Pectin and the like.
  • acidulant math, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like can be used.
  • the acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.
  • thickening agent examples include suspending agents, sedimentation agents, gel-forming agents, bulking agents and the like.
  • the food composition of the present invention may contain physiologically active substances or minerals which are known in the art and which are stable as a food additive in addition to the above-mentioned food additives in order to supplement and supplement functional and nutritional properties.
  • physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E and vitamin B12, tocopherol, dibenzoyl thiamine, etc.
  • minerals include calcium preparations such as calcium citrate, magnesium stearate , Iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc and the like.
  • the food composition of the present invention may contain an appropriate amount of the above-mentioned food additives according to the product type so as to achieve the purpose of addition thereof.
  • composition of the present invention can be identified as a pharmaceutical composition.
  • the pharmaceutical composition of the present invention may be prepared into oral formulations or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient.
  • the route of administration may be any suitable route including local routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucosal tissues, and combinations of two or more routes may be used.
  • An example of a combination of two or more routes is a combination of two or more formulations of the drug according to the route of administration, for example, one drug is administered intravenously and another drug is administered via a local route.
  • Pharmaceutically acceptable carriers are well known in the art depending on the route of administration and formulation, and specific reference may be made to the pharmacopoeia of each country, including the " Korean Pharmacopoeia ".
  • the pharmaceutical composition of the present invention When the pharmaceutical composition of the present invention is prepared into an oral formulation, it may be formulated into powder, granules, tablets, pills, sugar tablets, capsules, solutions, gels, syrups, suspensions, wafers And the like.
  • suitable carriers include starches such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol and xylitol, corn starch, potato starch and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Hydroxypropylmethylcellulose and the like; polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol Serol, and the like.
  • suitable binders include starch, magnesium aluminum silicate, starch pellets, gelatin, methyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax and the like.
  • disintegrating agent examples include starch, methylcellulose, magnesium stearate, magnesium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, Agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like.
  • diluent examples include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine and the like.
  • the pharmaceutical composition of the present invention When the pharmaceutical composition of the present invention is prepared into a parenteral dosage form, it may be formulated in the form of an injection, transdermal drug delivery, nasal aspirate and suppository together with a suitable carrier according to methods known in the art.
  • a suitable carrier aqueous isotonic solutions or suspensions may be used.
  • PBS phosphate buffered saline
  • isotonic solution such as 5% dextrose
  • transdermal preparation it can be formulated in the form of ointments, creams, lotions, gels, external liquids, pastes, liniments, and air-lozenges.
  • Nasal inhalers may be formulated in the form of aerosol sprays using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, witepsol, tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.
  • suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc.
  • the preferred dosage of the pharmaceutical composition of the present invention is 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / day, depending on the patient's condition, body weight, sex, age, / kg < / RTI > The administration can be carried out once or several times a day. Such dosages should in no way be construed as limiting the scope of the invention.
  • the composition of the present invention can be identified as a cosmetic composition.
  • a cosmetic composition its use can be understood as application to skin irritation or suppression of skin troubles such as erythema, dryness, burning, erythema, drying and bleeding caused by abnormal immunity.
  • the cosmetic composition may be classified into any product category according to the use of the cosmetic composition.
  • the cosmetic composition may contain functional cosmetic products having applications such as improvement of skin troubles and improvement of atopic dermatitis, General cosmetics, and the like.
  • the product form include a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing oil, powder foundation, emulsion foundation, wax Foundation, spray, and the like.
  • it may be a form of flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
  • the cosmetic composition of the present invention may contain, in addition to its active ingredient, conventional additives such as stabilizers, solubilizing agents, surfactants, vitamins, colorants and antioxidants, and carriers commonly used in cosmetic compositions.
  • conventional additives such as stabilizers, solubilizing agents, surfactants, vitamins, colorants and antioxidants, and carriers commonly used in cosmetic compositions.
  • the formulation of the present invention is a paste, a cream or a gel
  • an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component .
  • lactose When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component.
  • a spray in particular, / Propane or dimethyl ether.
  • a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component.
  • a solvent e.g., water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid esters of sorbitan, and the like.
  • a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar, etc. may be used.
  • the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.
  • the cosmetic composition of the present invention can be produced according to a method for producing a cosmetic composition which is usually carried out in the art, except that it contains an active ingredient showing atopic dermatitis improving activity or skin immunity hypersensitivity inhibitory activity.
  • an anti-inflammatory composition As described above, according to the present invention, it is possible to provide an anti-inflammatory composition, an anti-allergic composition and a composition for improving atopic dermatitis using the extracts of Ganoderma lucidum and Horns Cyprinus mellifera.
  • the anti-allergic composition and the composition for improving atopic dermatitis of the present invention can be commercialized into foods, cosmetics, medicines, etc. for use in anti-inflammatory use, antiallergic use, improvement of atopic dermatitis or suppression of skin irritation immune response.
  • Fig. 1 shows the results of cytotoxicity evaluation of mouse macrophage cell line of hoe saengmyung mackerel, ganoderma lucidum extract and mixture thereof.
  • FIG. 2 shows the results of evaluating the effect of hoe saengmyung mackerel, ganoderma lucidum extract, and mixtures thereof on cell viability of mouse macrophage cell line by LPS stimulation.
  • FIG. 3 shows the results of evaluating the effects of Hornsby moth, Liliaceae extract, and mixtures thereof on NO production of mouse macrophage cell line by LPS stimulation.
  • FIGS. 4 to 6 are the results of evaluating the effects of the hornbill venom, phlegmon extract and mixtures thereof on the secretion of inflammatory cytokines (IL-1 ⁇ , IL-6 and TNF- ⁇ ) of mouse macrophages by LPS stimulation.
  • IL-1 ⁇ , IL-6 and TNF- ⁇ inflammatory cytokines
  • FIG. 7 shows the ⁇ -hexosaminidase assay results of inhibiting the degranulation inhibition of the hoe saengmyung mackerel, ganoderma lucidum extract and mixtures thereof.
  • FIG. 8 shows RT-PCR results on the effect of the 5: 5 mixture of hornblende extract and gentian extract on the expression of inflammatory cytokines.
  • FIG. 9 is a western blot result of the effect on the expression of NF-.kappa.B, a transcription factor of inflammatory cytokine, of a mixture of Horns Cyprinus mellifera and a 5: 5 mixture of moxibustion extracts.
  • FIGS. 10 and 11 are the results of evaluating the effect of a 5: 5 mixture of the extract of hoe saengmyung mackerel and its moth extract on clinical symptoms of the skin in an atopic dermatitis animal model.
  • FIGS. 12 and 13 are the results of evaluating the effect of a 5: 5 mixture of hornblende extract and its gentle extract on ear edema in an atopic dermatitis animal model.
  • FIGS. 14 and 15 are the results of evaluating the effect of the mixture of hoe saengmyung mackerel extract and phytotoxic extract 5: 5 on moisture change in an atopic dermatitis animal model.
  • FIGS. 16 and 17 are the results of evaluating the effects of a 5: 5 mixture of hornblende extract and its sensory extract on skin itching symptoms in an atopic dermatitis animal model.
  • FIGS. 18 and 19 are the results of evaluating the effect of a 5: 5 mixture of hornblende extract and its sensory extract on the spleen size change in an atopic dermatitis animal model.
  • FIGS. 20 and 21 are the results of evaluating the effect of a 5: 5 mixture of hornblende extract and its gentle extract on the lymph node size in an atopic dermatitis animal model.
  • FIG. 22 shows the results of evaluating the effect of the mixture of hoe saengmyung mackerel extract and moxibustion extract 5: 5 on the blood levels of IgE, IgG1, and IgG2a in an animal model of atopic dermatitis.
  • Ecklonia cava (outpost) was collected from July to August 2016 in the coast of Jeju Island, Korea and washed with water to remove salt and other impurities. After washing with water, 20 g of ethanol was added to 70% ethanol for 24 hours, and filtered. The filtrate was concentrated under reduced pressure and lyophilized to prepare a powdery extract (ECE, Ecklonia cava ethanol extract).
  • Example 1 The ECE of Example 1 and the SHE of Example 2 were changed to 0:10 (SHE alone), 2: 8, 4: 6, 5: 5, 6: 4, 8: ECE alone) (ECE: SHE) (mixture: MES).
  • the macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO 2 incubator. 1 ⁇ 10 5 cells per well were dispensed in 96-well plates and cultured for 16 hours. After incubation for 24 h at 37 ° C in a 5% CO 2 incubator, 50 ⁇ l of MTT (2 mg / ml) was added to each well. After incubation at 37 ° C in a 5% CO 2 incubator for 2 hours, the solution was removed and treated with 150 L of DMSO to dissolve the Formazan precipitate formed and the absorbance was measured at 540 nm using ELISA Was measured.
  • Fig. Figure 1 shows that ECE, SHE and mixtures thereof show no cytotoxicity in particular.
  • the macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO 2 incubator. 1 ⁇ 10 5 cells per well were dispensed in 96-well plates and cultured for 16 hours. After 1 hour, LPS (lipopolysaccharide, 1 ⁇ g / mL) inducing inflammation was treated and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. 50 ⁇ l of MTT (2 mg / ml) Respectively. After incubation at 37 ° C in a 5% CO 2 incubator for 2 hours, the solution was removed and treated with 150 ⁇ l of DMSO to dissolve the Formazan precipitate and the absorbance was measured at 540 nm using ELISA Was measured.
  • LPS lipopolysaccharide, 1 ⁇ g / mL
  • the macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO 2 incubator. 1 ⁇ 10 5 cells per well were dispensed in 96-well plates and cultured for 16 hours. After 1 hour, LPS (lipopolysaccharide, 1 ⁇ g / mL) inducing inflammation was treated and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours.
  • LPS lipopolysaccharide, 1 ⁇ g / mL
  • the macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO 2 incubator. 1 ⁇ 10 5 cells per well were dispensed in 96-well plates and cultured for 16 hours. After 1 hour, the cells were treated with LPS (lipopolysaccharide, 1 / / mL) inducing inflammation and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. IL-1 ⁇ , IL-6 And TNF- [alpha] cytokines were analyzed using real-time PCR. The samples used here were ECE, SHE and mixtures of 5: 5 and 8: 2.
  • ⁇ -hexosaminidase The secretion of ⁇ -hexosaminidase was measured in order to determine inhibitory effects of BMCMC on detachment, hornbill, hornblende and hornblende.
  • Cells (5 ⁇ 10 5 cells) were sensitized with anti-DNP-IgE (1 ⁇ g / ml) for 1 hour after preincubation at 37 ° C. for 2 hours with various concentrations of ECE, SHE and MES.
  • tyrode buffer (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose and 0.1% BSA, pH 7.4) and incubated with DNP-BSA ) At 37 < 0 > C for 1 hour. After obtaining 30 ⁇ l of supernatant, the cells were lysed by tyroide buffer containing 1% Triton X-100.
  • the supernatant (5 ⁇ l) and the dissolved cells (5 ⁇ l) were incubated with 50 ⁇ l of substrate solution (2 mM p-nitrophenyl-N-acetyl-D-glucosaminide, pH 4.5) in a 96- And reacted at 37 ° C.
  • the reaction was terminated by treating 150 ⁇ l of 0.2 M Glycine-NaOH buffer (pH 10.7), and the absorbance was measured at 405 nm using a microplate reader.
  • the percentage of ⁇ -hexosaminidase secreted from the supernatant was calculated by the following formula.
  • ⁇ -hexosaminidase% [absorbance of supernatant / absorbance of supernatant + absorbance of cell] ⁇ 100
  • FIG. Figure 7A shows that the treated samples significantly inhibited the secretion of ⁇ -hexosaminidase, an indicator factor of degranulation, and showed the best activity of the 5: 5 mixture in particular.
  • 7B shows that the 5: 5 mixture inhibits the secretion of? -Hexosaminidase in a concentration-dependent manner.
  • a 5: 5 mixture with the best activity was used as a sample.
  • BMCMC cells were treated with various concentrations of BMCMC for 2 hours and sensitized with anti-DNP-IgE (1 ⁇ g / ml) for 4 hours.
  • Cells were washed with tyrode buffer and stimulated with DNP-BSA (100 ng / ml) for 24 h at 37 ° C. Twenty-four hours later, the supernatant was removed, and triazol reagent was added to the cells. After adding chloroform, the cells were vortexed for 10 seconds and centrifuged at 12,000 rpm for 15 minutes. The supernatant was removed and mixed with isopropanol.
  • CDNA was synthesized using a cDNA synthesis kit, and the cDNAs were denatured at 45 ° C for 30 minutes and at 94 ° C for 30 seconds using the cytokine and chemokine primer and RT-PCR kit shown in Table 2, After 30 seconds of annealing, the cycle of extraction at 72 ° C for 30 seconds was repeated 32 times and the final extension was performed at 72 ° C for 5 minutes. Each PCR product was loaded on 1.5% agarose gel and analyzed by electrophoresis for 30 minutes at 100V. The results are shown in Fig.
  • FIG. 8 shows that the 5: 5 mixture was increased in concentration-dependent manner by treatment with anti-DNP-IgE and DNP-BSA, and IL-1 ⁇ , IL-4, IL-5, IL-6, TNF- Inhibit the expression of cytokines and suppress the expression of TSLP and TARC, which are known to increase in atopic dermatitis.
  • BMCMC cells were cultured for 2 hours at various concentrations and sensitized with anti-DNP-IgE (1 ⁇ g / ml) for 4 hours. Cells were washed with tyrode buffer and stimulated with DNP-BSA (100 ng / ml) for 30 min at 37 ° C. After 30 minutes, the supernatant was removed, and lysis buffer was added to the cells. The vortexing was carried out for 6 times for 10 minutes. The supernatant was centrifuged at 14,000 rpm for 10 minutes at 4 ° C and proteins were quantified using BCA kit.
  • a mixture of a mixture of a mixture of a mixture of a hoe saengmyungbom extract (SHE) and a 5: 5 weight ratio of a hawthorn extract and a hornblende extract, which exhibited excellent activity in the anti-inflammatory and anti- : SHE 5: 5) was used as a sample and the sample was orally administered to an animal model in which atopic dermatitis was induced, and clinical symptoms and skin itching degree were evaluated.
  • 1% DNCB (Dinitrochlorobenzene) was applied to Nc / Nga mice deprived of the back region to induce atopic dermatitis first.
  • 1% DNCB and AD ointment 100 mg were applied to the depilated area, Induced atopic dermatitis.
  • oral administration was performed daily for 2 weeks and clinical analysis and scratching analysis were performed weekly.
  • the animals were sacrificed and the ear thickness, transepidermal water loss (TEWL), water and pH were measured and the blood, spleen, and lymph nodes were separated and analyzed.
  • TEWL transepidermal water loss
  • ear thickness of each individual was measured at 4th week. After animals were anesthetized, the right ear and left ear were divided into three parts, respectively, and caliper was used to measure the thickness.
  • Transepidermal water loss Loss of water through the skin is caused by evaporation of skin moisture and perspiration. Among these, transdermal water loss is caused by passive diffusion of moisture from the inside of the body to the skin, It is called water loss. To evaluate skin dryness, which is a typical clinical symptom of atopic dermatitis, transdermal water loss was measured at the 4th week of the animal experiment. After the animals were anesthetized, the atrophy-induced back area was measured for 30 seconds using a transcutaneous water loss measuring device (AquaFlux, Biox).
  • Moisture was measured at 4th week after the end of the animal experiment.
  • Skin-o-mat Cosmetic Medical Beauty
  • Atopic dermatitis reactions cause a variety of responses in the immune system, primarily causing hypertrophy of the immune system. Therefore, the spleen and lymph node weights were measured in order to observe the effect of the 5:
  • lymph node size The results for lymph node size are shown in Figs. 20 and 21, which alleviated the hypertrophy of both cLN (superficial cervical lymph node), aLN (axillary lymph node), bLN (brachial lymph node) and dLN .

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Abstract

La présente invention concerne une composition anti-inflammatoire, antiallergène et améliorant la dermatite atopique utilisant un extrait de Sargassum horneri ou un extrait d'Ecklonia veine, la composition ayant été développée en confirmant que l'extrait de Sargassum horneri ou l'extrait d'Ecklonia veine présente une activité anti-inflammatoire et une activité antiallergique dans des essais cellulaires et une activité d'amélioration de la dermatite atopique dans des essais chez l'animal.
PCT/KR2018/012234 2017-10-18 2018-10-17 Composition anti-inflammatoire, antiallergène et améliorant la dermatite atopique utilisant un extrait de sargassum horneri ou un extrait d'ecklonia veine WO2019078606A2 (fr)

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CN117122059A (zh) * 2023-08-29 2023-11-28 中国海洋大学 一种抗过敏和抗炎症海藻多酚的制备方法与应用

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